Combinations of Radiotherapy with Vaccination and Immune Checkpoint Inhibition Differently Affect Primary and Abscopal Tumor Growth and the Tumor Microenvironment

Round 1

Reviewer 1 Report

“Combinations of radiotherapy with vaccination and immune  checkpoint inhibition differently affect primary and abscopal tumor growth and the tumor microenvironment” by Michael Rückert et al.

 

This manuscript focuses on evaluating the combinatorial antitumor effect of radiotherapy, cancer vaccine and immune checkpoint inhibitors in B16 melanoma and TS/A mammary carcinoma mouse models. Using two different hypofractionated radiotherapeutic schedules, whole tumor cell vaccines generated with high hydrostatic pressure, immune check point inhibitors and analyzing the local and systemic immune responses, the authors found that tumor vaccines work systemically, but only on previously hypofractionated irradiated tumors. They also revealed that abscopal responses to (radiotherapy plus anti-PD1) were abrogated if the number of fractions of radiotherapy was too high, essentially driven by the upregulation of further immune checkpoint ligands. The authors concluded that autologous whole tumor cell-based vaccines induce antitumor effects, but only if the tumor was previously irradiated. However, these vaccines do not increase abscopal antitumor immunity of radiotherapy plus checkpoint inhibition. Altogether, these suggest that the immunotherapies profit from the radiotherapy-induced tumor microenvironment modulations but instead of pure infiltration of immune cells, it is rather a functional alteration of pre-existing or newly infiltrating immune cells that mediate the improved anti-tumor immune response. 

 

Abscopal effect is an interesting phenomenon in radiobiology that causes activation of immune system toward malignant cells. Traditionally, this phenomenon was known as a suppressor of non-irradiated tumors or metastasis. However, it can be used as a stimulator of the immune system against primary tumors during radiotherapy. Some studies have shown that radiotherapy or immunotherapy administered alone have low efficiency for growing tumor, however, their combination may have a more potent antitumor immunity. Combination therapy with least side effect is an interesting and clinically important strategy to achieving complete tumor control. For this aim, it is important to induce abscopal effect in primary tumors, and also use appropriate approaches to target the mechanisms involved in the exhaustion of effector immune cells. Thus, this manuscript presents important and novel results of interesting combinational therapeutic approach and evaluate the mechanisms of its antitumor potential. Furthermore, the offered antitumor combination is known as the best choice among various strategies for radioimmunotherapy. However, there is the need for other strategies to improve the duration of immune system’s activity within the tumor microenvironment. And this is the main focus of the manuscript.

 

The main strengths of the manuscript is not just an introduction of a fascinating combinational approach, but a comprehensive analysis of immune cells – both local (intratumoral) and systemic (blood), which provide an opportunity for better understanding of specific immune responses and better evaluation of abscopal pathways.

 

Overall, all results are well presented, perfectly discussed, and very well support the authors’ conclusions. All statistical considerations are in place and all required controls were tested and verified. All figure legends are informative and correct. M&M are very well described and quite informative. Discussion is outstanding and very logically organized but might be too long for easy reading. All references are appropriate. There are no major concerns or suggestions, and only a few minor comments should be addressed.

 

Minor comments:

• Although the abscopal is very briefly introduced in Background, it’s description should be slightly improved since many readers may be not familiar with radiobiology
• The phrase “concentration of T cells” does not sound (page 11-358 and Fig.4 legend)
• In the description of cell cycle analysis methodology, the concentrations of PI and RNase should be stated
• Concentrations of all components of tumor lysis buffer should be included in M&M
• Since the authors use “pDC” abbreviation, it might be better to use “cDC” abbreviation instead of “DC”. However, this is just a suggestion.

 

 

Author Response

Reviewer 1

“Combinations of radiotherapy with vaccination and immune  checkpoint inhibition differently affect primary and abscopal tumor growth and the tumor microenvironment” by Michael Rückert et al.

 

This manuscript focuses on evaluating the combinatorial antitumor effect of radiotherapy, cancer vaccine and immune checkpoint inhibitors in B16 melanoma and TS/A mammary carcinoma mouse models. Using two different hypofractionated radiotherapeutic schedules, whole tumor cell vaccines generated with high hydrostatic pressure, immune check point inhibitors and analyzing the local and systemic immune responses, the authors found that tumor vaccines work systemically, but only on previously hypofractionated irradiated tumors. They also revealed that abscopal responses to (radiotherapy plus anti-PD1) were abrogated if the number of fractions of radiotherapy was too high, essentially driven by the upregulation of further immune checkpoint ligands. The authors concluded that autologous whole tumor cell-based vaccines induce antitumor effects, but only if the tumor was previously irradiated. However, these vaccines do not increase abscopal antitumor immunity of radiotherapy plus checkpoint inhibition. Altogether, these suggest that the immunotherapies profit from the radiotherapy-induced tumor microenvironment modulations but instead of pure infiltration of immune cells, it is rather a functional alteration of pre-existing or newly infiltrating immune cells that mediate the improved anti-tumor immune response. 

 

Abscopal effect is an interesting phenomenon in radiobiology that causes activation of immune system toward malignant cells. Traditionally, this phenomenon was known as a suppressor of non-irradiated tumors or metastasis. However, it can be used as a stimulator of the immune system against primary tumors during radiotherapy. Some studies have shown that radiotherapy or immunotherapy administered alone have low efficiency for growing tumor, however, their combination may have a more potent antitumor immunity. Combination therapy with least side effect is an interesting and clinically important strategy to achieving complete tumor control. For this aim, it is important to induce abscopal effect in primary tumors, and also use appropriate approaches to target the mechanisms involved in the exhaustion of effector immune cells. Thus, this manuscript presents important and novel results of interesting combinational therapeutic approach and evaluate the mechanisms of its antitumor potential. Furthermore, the offered antitumor combination is known as the best choice among various strategies for radioimmunotherapy. However, there is the need for other strategies to improve the duration of immune system’s activity within the tumor microenvironment. And this is the main focus of the manuscript.

 

The main strengths of the manuscript is not just an introduction of a fascinating combinational approach, but a comprehensive analysis of immune cells – both local (intratumoral) and systemic (blood), which provide an opportunity for better understanding of specific immune responses and better evaluation of abscopal pathways.

 

Overall, all results are well presented, perfectly discussed, and very well support the authors’ conclusions. All statistical considerations are in place and all required controls were tested and verified. All figure legends are informative and correct. M&M are very well described and quite informative. Discussion is outstanding and very logically organized but might be too long for easy reading. All references are appropriate. There are no major concerns or suggestions, and only a few minor comments should be addressed.

First we want to thank the reviewer for the positive feed-back.

 

Minor comments:

• Although the abscopal is very briefly introduced in Background, it’s description should be slightly improved since many readers may be not familiar with radiobiology

Answer: We agree with the reviewer’s suggestion an have complemented this information in the manuscript as follows:

In the introduction we describe the abscopal effects in lines 47 – 61. We now added the additional information how the local effects spread systemically: “Dendritic cells (DCs) take up the tumor antigen, transport it to the draining lymph node and present it there to T cells, which are subsequently released into the periphery”.

Additionally we added to the abstract (line 30): “….abscopal anti-tumor responses, namely those to non-irradiated tumors, ….”

• The phrase “concentration of T cells” does not sound (page 11-358 and Fig.4 legend)

Answer: We apologize if the wording might sound inappropriate. We wanted to avoid writing “infiltration” as for example more T cells per gram of tumor does not necessarily need to be a result of infiltration, but might be due to a decreased tumor mass. Therefore, we intentionally used “concentration” as we think it best reflects the conditions.

• In the description of cell cycle analysis methodology, the concentrations of PI and RNase should be stated

Answer: We have added the concentrations on page 4 line 186

• Concentrations of all components of tumor lysis buffer should be included in M&M

Answer: We have added the concentrations on page 5 lines 202-205

• Since the authors use “pDC” abbreviation, it might be better to use “cDC” abbreviation instead of “DC”. However, this is just a suggestion.

Answer: Thanks for this note. In the context of our immune phenotyping results, if we use “DC”, we refer to all MHC-II+, CD11c+ cells. “cDC1s” refer to the subset of “DCs”, which are CD11b- and “pDCs” are gated totally different as CD11b-, Ly6G-, PDCA-1+, Ly6C+. Thus, these terms cannot be used interchangeably.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The authors evaluate a therapeutic approach by combing radiotherapy, PD-1 blockade and cancer vaccination. Specific major comments are provided below.

 1) In Figures 1, 2 and 9, authors should compare the tumor growth in combination treatment against single agents, not just against the control group. 

2) There is no statistical analysis in Figure 2. The authors must perform rigorous statistical analysis to state that they observed similar benefits. 

3) The authors should include the color scheme for each treatment in all figures, not just figure 1.

4) The authors should mention their gating strategies Figure S1-3 in the main text. 

5) The authors have too many figures with similar information. Figures 1 and 2 can be combined as one figure. Figures 4,5, and 6 need to be merged together. Also, Figures 10 and 11 should be combined as one figure. 

Minor point:

Line 25 on page 1, "synergize to induce tumor growth retardation" should change to "synergize to retard the tumor growth"

 

 

 

 

 

 

 

Author Response

Reviewer 2

The authors evaluate a therapeutic approach by combing radiotherapy, PD-1 blockade and cancer vaccination. Specific major comments are provided below.

 1) In Figures 1, 2 and 9, authors should compare the tumor growth in combination treatment against single agents, not just against the control group. 

Answer: We thank the reviewer for this note and want to clarify as follows: we already have previously shown that HHP vaccines alone do not have any effect on the tumor growth (Seitz et al., 2019). Further, B16 tumors are known to be refractory to immune checkpoint inhibition alone (Liang et al., 2018; Sharabi et al., 2015).

2) There is no statistical analysis in Figure 2. The authors must perform rigorous statistical analysis to state that they observed similar benefits. 

Answer: We apologize for not having included the statistical tests. We initially had only shown one representative experiment. Now we have included the data of a second independent experiment, performed the same statistical analyses as in figure 1 and indicated the number of animals, as also requested by the editor.

3) The authors should include the color scheme for each treatment in all figures, not just figure 1.

Answer: We have now used the same color scheme for all in vivo experiments (figure1 – 8 and 10f).

4) The authors should mention their gating strategies Figure S1-3 in the main text. 

Answer: We thank the reviewer for this advice and included “The different cell types were identified according to the gating strategies shown in Figure S1-3.” on page 9 lines 314-315.

5) The authors have too many figures with similar information. Figures 1 and 2 can be combined as one figure. Figures 4,5, and 6 need to be merged together. Also, Figures 10 and 11 should be combined as one figure. 

Answer: We followed the editor’s suggestion and merged figure 4 and 5.

Minor point:

Line 25 on page 1, "synergize to induce tumor growth retardation" should change to "synergize to retard the tumor growth"

Answer: We agree with the reviewer and changed the phrase accordingly on page 1 line 25

 

Editor Decision

This is a very interesting manuscript providing important data regarding combinatorial radiotherapy approaches. The data is comprehensive and rigorously evaluated with well designed experimental studies. The number of animals used per cohort in Figure 2 should be included for clarity. There is a considerable amount of data in the manuscript and the Figures themselves are highly detailed and should remain as they are. However, it is possible that Figure 4 and 5 could be merged together, and this should be considered.

Answer: We want to thank the reviewers and the editor for the thorough judging of the data, for giving us the possibility of revisions and for the overall positive feedback. We merged figures 4 and 5 and changed the figure numbering in the text accordingly. Further, we included the statistical tests in figure 2. We initially had only shown one representative experiment. Now we have included the data of a second independent experiment, performed the same statistical analyses as in figure 1 and indicated the number of animals. Due to the valuable comments, we think the manuscript has improved and is now suitable for publication in Cancers.

 

 

References

Liang, Y., Tang, H., Guo, J., Qiu, X., Yang, Z., Ren, Z., . . . Fu, Y.-X. (2018). Targeting IFNα to tumor by anti-PD-L1 creates feedforward antitumor responses to overcome checkpoint blockade resistance. Nature Communications, 9(1). doi:10.1038/s41467-018-06890-y

Seitz, C., Rückert, M., Deloch, L., Weiss, E. M., Utz, S., Izydor, M., . . . Frey, B. (2019). Tumor Cell-Based Vaccine Generated With High Hydrostatic Pressure Synergizes With Radiotherapy by Generating a Favorable Anti-tumor Immune Microenvironment. Front Oncol, 9, 805. doi:10.3389/fonc.2019.00805

Sharabi, A. B., Nirschl, C. J., Kochel, C. M., Nirschl, T. R., Francica, B. J., Velarde, E., . . . Drake, C. G. (2015). Stereotactic Radiation Therapy Augments Antigen-Specific PD-1–Mediated Antitumor Immune Responses via Cross-Presentation of Tumor Antigen. Cancer Immunology Research, 3(4), 345-355. doi:10.1158/2326-6066.Cir-14-0196

 

Author Response File: Author Response.docx